Quant it ribogreen rna reagent and kit

Total RNAs were extracted from cell lysates with SV96 Total RNA Isolation System (Promega, WI) according to manufacturer's protocol. Total RNAs extracted from each treatment group at the same time point were pooled into one tube for the downstream processes. A total of six RNA samples were obtained representing two treatments at three time points. Quantity of the pooled RNA samples was determined with Quant-iT RiboGreen RNA Reagent and Kit (Molecular Probes, OR), while the quality and integrity of RNA were confirmed with spectrophotometry and formaldehyde 1% agarose gel electrophoresis. All total RNA samples possessed A260/A280 ratio > 1.8 and the 28S:18S rRNA intensity ratio of 1.76 estimated with Nanodrop 2000 (Thermo Scientific, DE) and Quantity One Analysis Software (Bio-Rad, CA) respectively.

DNA isolation from tail tip biopsies was carried out by standard protocols or using the Qiagen DNA Blood and Tissue Kit (Qiagen, Hilden, Germany). RNA was isolated from cells and tissues using the RNeasy Mini Kit and from fatty tissues using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany). For all RNA isolations DNAse digestion was routinely performed. Concentrations were determined using a NanoDrop spectrophotometer (Peqlab, Erlangen, Germany) and the Quant-iT RiboGreen RNA reagent and kit (Life Technologies, Darmstadt, Germany). Reverse Transcription was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For routine PCR we used recombinant Taq DNA polymerase and for cloning procedures AccuPrime Pfx DNA polymerase (Life Technologies, Darmstadt, Germany). For quantitative real-time PCR we used LightCycler FastStart DNA Master SYBR Green I on a Roche Light Cycler 1.5 (Roche, Rotkreuz, Switzerland). Oligonucleotides and PCR conditions are given in dataset S4.

FFPE tumor ribbons from patients with Ewing sarcoma were deparaffinized with Hemo-De, tissue was washed twice in 100% ethanol, and allowed to air dry for 45 minutes. Tissue was digested and RNA was isolated following the Recoverall Total Nucleic Acid Isolation protocol (Ambion Life Technologies). RNA was isolated from cell lines using QIAGEN's RNeasy Mini protocol with QIAshredder (QIAGEN). RNA was treated with DNase using the TURBO DNA-free Kit (Ambion Life Technologies) and quantified using Quant-iT Ribogreen RNA Reagent and Kit (Invitrogen).

The whole full thickness rumen wall samples were shipped to CSIRO’s FD McMaster Laboratory at Chiswick, Armidale, Australia and transferred into RNALater^®-ICE Frozen Tissue Transition Solution (Ambion^®) and stored at −20 °C. RNA was extracted from the full thickness of ~200 mg of ventral rumen tissue using the Qiagen RNeasy^® Midi Kit with on-column DNase digestion. The average RNA concentration measured using a Quant-iT™ RiboGreen^® RNA reagent and kit (Invitrogen™) on a fluorescent plate reader (excitation 485 nm and emission 538 nm) was 333.9 ± 143.4 (SD) ng/μl. Samples of ~4 μg of total RNA were rRNA depleted using the Ribo-Zero™ Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre^®) and purified using a Qiagen RNeasy^® MinElute Cleanup Kit. 12 μl rRNA depleted RNA was sent to the Ramaciotti Genomics Centre, The University of New South Wales, Australia. RNA Quality was checked and stranded libraries were prepared using SureSelect™ stranded RNA sample preparation kit (Agilent Technologies) at the sequencing centre. RNA sequencing was performed as 100 base paired-end (PE) reads in two lanes of an Illumina HiSeq2000 instrument housed at the Ramaciotti Genomics Centre. Total reads per sample ranged from 15–20 (single reads) or 30–40 million (PE) per sample. RNA sequencing results were checked for quality using FastQC at the sequencing center.